Dose-Dependent Genome-Wide DNA Methylation Remodeling by Metformin Modulates Doxorubicin Sensitivity in Cardiac Cells

Figures

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  Figure 1. Metformin (MET) pre-treatment shifts doxorubicin (DOX) dose–response curves in H9c2 cardiomyoblast cells. (A) DOX alone, (B) DOX and 0.7 mM MET, (C) DOX and 1.4 mM MET, and (D) DOX and 2.8 mM MET.

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  Figure 2. Principal component analysis reveals dose-dependent genome-wide DNA methylation changes following chronic metformin exposure. Green circles represent control samples, purple squares represent low-dose MET-treated cells, and orange triangles represent high-dose MET-treated H9c2 cells.

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  Figure 3. Differential DNA methylation analysis identifies hyper- and hypomethylated regions across metformin treatment conditions. (A) Volcano plot of differentially methylated regions (DMRs) in high-dose MET versus control; (B) low-dose MET versus control; and (C) high-dose MET versus low-dose MET. The x-axis represents Δβ (difference in methylation levels between groups), and the y-axis represents −log10 FDR-adjusted p-values. Each point represents a DMR, with significant regions defined by FDR < 0.05 and the applied Δβ threshold.

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  Figure 4. Hierarchical clustering of differentially methylated regions demonstrates dose-dependent epigenetic divergence between treatment groups. Hierarchical clustering heatmap of differentially methylated 1 kb-genomic tiles in H9c2 cardiomyoblast cells treated with low-dose and high-dose metformin compared with untreated controls. Rows represent genomic regions, and columns represent individual samples. Methylation levels are displayed as scaled β-values (z-score), with red indicating higher methylation and blue indicating lower methylation. Unsupervised clustering reveals a clear separation of samples according to treatment group, demonstrating a dose-dependent epigenetic reprogramming effect of metformin on the cardiac DNA methylation landscape.

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  Figure 5. Enrichment of ATP-dependent chromatin remodeling complexes associated with low-dose metformin treatment. The schematic illustrates the major families of ATP-dependent chromatin remodeling complexes (CRCs), including ISWI, CHD (NuRD), SWI/SNF (BAF/PBAF), and INO80/SWR, which regulate chromatin structure through nucleosome sliding, spacing, ejection, histone dimer removal, and histone variant exchange. Pathway enrichment analysis was performed using DAVID [14].

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  Figure 6. Chromatin remodeling pathways enriched following high-dose metformin-induced epigenetic remodeling. The schematic highlights key ATP-dependent chromatin remodelling families and their regulatory roles in chromatin accessibility and transcriptional control. Pathway enrichment analysis was performed using DAVID [14].